Photoinduced electron-transfer alpha-deoxygenation of aldonolactones. Efficient synthesis of 2-deoxy-D-arabino-hexono-1,4-lactone

A photoinduced electron-transfer (PET) reaction was used for the deoxygenation at C-2 of aldonolactones derivatized as 2-O-[3-(trifluoromethyl)benzoyl] or benzoyl esters. By irradiation of different D-galactono- and D-glucono-1,4-derivatives, with a 450W lamp, using 9-methylcarbazole as photosensitizer, the corresponding 2-deoxy-D-lyxo- and 2-deoxy-D-arabino-hexono-1,4-lactones were efficiently obtained.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

Total synthesis and identification of two diastereoisomers of 1-methyl-2-[N-(p-tolylsulfonyl)-2-pyrrolidinyl]ethyl beta-L-fucopyranoside

The reaction of a racemic mixture of (2R,2'S)- and (2S,2'R)-N-(p-tolylsulfonyl)-2-pyrrolidinyl-2-propanol, prepared from (S)-proline, with 2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl trichloroacetimidate led to both diastereoisomers of the title compound after O-deacetylation.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

l-Lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative l-lactate oxidase localized in the intermembrane space

In order to ascertain whether and how mitochondria can produce hydrogen peroxide (H₂O₂) as a result of l-lactate addition, we monitored H₂O₂ generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H₂O₂ is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent l-lactate oxidase restricted to the intermembrane space. The l-lactate oxidase reaction shows a hyperbolic dependence on l-lactate concentration and is inhibited by NAD⁺ in a competitive manner, being the enzyme different from the l-lactate dehydrogenase isoenzymes as shown by their pH profiles.     

Inhibition of the D-fructose transporter protein GLUT5 by fused-ring glyco-1,3-oxazolidin-2-thiones and -oxazolidin-2-ones

The glucose transporter 5 (GLUT5)-a specific D-fructose transporter-belongs to a family of facilitating sugar transporters recently enlarged by the human genome sequencing. Prompted by the need to develop specific photolabels of these isoforms, we have studied the interaction of conformationally locked D-fructose and L-sorbose derived 1,3-oxazolidin-2-thiones and 1,3-oxazolidin-2-ones to provide a rational basis for an interaction model. The inhibition properties of the D-fructose transporter GLUT5 by glyco-1,3-oxazolidin-2-thiones and glyco-1,3-oxazolidin-2-ones is now reported. In vitro, the fused-rings systems tested showed an efficient inhibition of GLUT5, thus bringing new insights on the interaction of D-fructose with GLUT5.     

Microwave-enhanced reductive amination via Schiff's base formation for block copolymer synthesis

Reductive amination via Schiff's base formation is a widely used reaction for laboratory and industrial applications ranging from protein immobilization to nanoparticle synthesis. One major limitation of this reaction is the slow kinetics and hence, it can take several days for the reaction to reach completion. Here we demonstrate that electromagnetic microwave can be used to accelerate the rate of reduction amination. To demonstrate proof of concept, we utilized the reductive amination between reducing end of dextran and primary amine from N-Boc-ethylenediamine as a model reaction. The reaction was conducted at room temperature to demonstrate that the enhancement was mainly due to electromagnetic effects of the microwave rather than thermal effects. We show that reductive amination reaction time was reduced from 72 h to 4 h using microwave irradiation. These results indicate non-thermal microwave effects to expedite reductive amination for synthesizing copolymers. The efficient conjugation of dextran using reductive amination provides an important tool for developing biocompatible copolymers using carbohydrates.     

Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate beta-lyase

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas flavipulchra NCIMB 2033(T)

An acidic polysaccharide was isolated from Pseudoalteromonas flavipulchra type strain NCIMB 2033(T) and found to consist of 6-deoxy-L-talose (L-6dTal), D-galactose and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The identities of the monosaccharides were ascertained by sugar analysis and 1D 1H and 13C NMR spectroscopy in conjunction with 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments, which enabled determination of the following structure of the trisaccharide repeating unit of the polysaccharide:-->3)-alpha-L-6dTalp4Ac-(1-->3)-beta-D-Galp-(1-->7)-alpha-Kdop-(2-->.     

A highly specific glyoxylate reductase derived from a formate dehydrogenase

A Glu141Asn mutant Paracoccus sp. 12-A formate dehydrogenase catalyzes marked glyoxylate reduction. Additional replacement of the His332-Gln313 pair with His-Glu, which is a consensus acid/base catalyst in D-hydroxyacid dehydrogenases, further improved the catalytic activity of the enzyme as to glyoxylate reduction through enhancement of the hydrogen transfer step in the catalytic process, slightly shifting the optimal pH for the reaction. On the other hand, the replacement induced no marked activity toward other 2-ketoacid substrates, and diminished the enzyme activity as to formate oxidation. Consequently, the formate dehydrogenase was converted to a highly specific and active glyoxylate reductase through only the two amino acid replacements.     

Synthesis of dopamine and l-DOPA-α-glycosides by reaction with cyclomaltohexaose catalyzed by cyclomaltodextrin glucanyltransferase

Dopamine-HCl and l-DOPA-α-glycosides were prepared by reaction with cyclomaltohexaose, catalyzed by Bacillus macerans cyclomaltodextrin glucanyltransferase. The reaction gave maltodextrins attached to dopamine and l-DOPA; the maltodextrins were trimmed by reactions with glucoamylase and β-amylase to produce α-glucosyl- and α-maltosyl-glycosides, respectively. The glucoamylase- or β-amylase-treated dopamine- and l-DOPA-α-glycosides were fractionated and purified by BioGel P-2 gel-filtration column chromatography and preparative descending paper chromatography. Analysis by MALDI-TOF mass spectrometry and one- and two-dimensional NMR showed that the purified glycosides of dopamine and l-DOPA were glycosylated at the hydroxyl groups of positions 3 and 4 of the catechol ring. The major product was found to be 4-O-α-glycopyranosyl l-DOPA, and it was shown to be more resistant to oxidative tolerance experiments, involving hydrogen peroxide and ferrous ion, than l-DOPA. l-DOPA-α-glycosides are possibly more effective substitutes for l-DOPA in treating Parkinson’s disease in that they are more resistant to oxidation and methylation, which renders l-DOPA ineffective and deleterious.     

The Hypocrea jecorina (syn. Trichoderma reesei) lxr1 gene encodes a d-mannitol dehydrogenase and is not involved in l-arabinose catabolism

The Hypocrea jecorina LXR1 was described as the first fungal l-xylulose reductase responsible for NADPH dependent reduction of l-xylulose to xylitol in l-arabinose catabolism. Phylogenetic analysis now reveals that LXR1 forms a clade with fungal d-mannitol 2-dehydrogenases. Lxr1 and the orthologous Aspergillus nigermtdA are not induced by l-arabinose but expressed at low levels during growth on different carbon sources. Deletion of lxr1 does not affect growth on l-arabinose and l-xylulose reductase activity remains unaltered whereas d-mannitol 2-dehydrogenase activities are reduced. We conclude that LXR1 is a d-mannitol 2-dehydrogenase and that a true LXR1 is still awaiting discovery.     

Synergism between hydrogen sulfide (H(2)S) and nitric oxide (NO) in vasorelaxation induced by stonustoxin (SNTX), a lethal and hypotensive protein factor isolated from stonefish Synanceja horrida venom

Stonustoxin (SNTX) is a 148 kDa, dimeric, hypotensive and lethal protein factor isolated from the venom of the stonefish Synanceja horrida. SNTX (10-320 ng/ml) progressively causes relaxation of endothelium-intact, phenylephrine (PE)-precontracted rat thoracic aortic rings. The SNTX-induced vasorelaxation was inhibited by L-N(G)-nitro arginine methyl ester (L-NAME), suggesting that nitric oxide (NO) contributes to the SNTX-induced response. Interestingly, D, L-proparglyglycine (PAG) and beta-cyano-L-alanine (BCA), irreversible and competitive inhibitors of cystathionine-gamma-lyase (CSE) respectively, also inhibited SNTX-induced vasorelaxation, indicating that H(2)S may also play a part in the effect of SNTX. The combined use of L-NAME with PAG or BCA showed that H(2)S and NO act synergistically in effecting SNTX-induced vasorelaxation.     

Transfer of nitric oxide from nitrovasodilators to free thiols--evidence of two distinct stages

Two distinct stages, which can be monitored spectrophotometrically, have been observed for the first time in multiple reactions between thiol, such as l-cysteine and some well-known nitrovasodilators, namely S-nitroso-N-acetyl-d,l-penicillamine (SNAP), S-nitrosoglutathione (GSNO), and S-nitrosocaptopril (SNOCap) in aqueous solution (in the presence of EDTA). The first part of the reaction occurs at stopped-flow time scale ( approximately 10(-2)s(-1)) in one single step and has been found to be transnitrosation; followed by a slow decomposition of the products of the transnitrosation reaction with the formation of a variety of nitrogen products. Reactivity with regard to the first stage occurs in the order GSNO>SNAP>SNOCap. The second stage shows first-order rate constants of the order 10(-4)s(-1), although some degree of complexity exists in elucidating an accurate rate equation with which a comparative study could be done.     

The structure of the antigenic polysaccharide produced by Eubactrium saburreum T15

The antigenic polysaccharide was obtained from the cell wall of Eubacterium saburreum strain T15 by trypsin digestion followed by gel permeation and ion-exchange chromatography. Its structure was determined using acid hydrolysis, methylation analysis, and 1D and 2D NMR spectroscopy. It contained L-threo-pent-2-ulose (Xul), D-fucose (Fuc), and D-glycero-D-galacto-heptose (Hep) in 2:3:3 ratio. Methylation analysis indicated an octasaccharide repeating-unit containing five branches. The 1H and 13C signals in NMR spectra of the sugar residues were assigned by COSY, HOHAHA, and HMQC 2D experiments, and the sequence of sugar residues in the repeating unit was determined by NOESY and HMBC experiments. The polysaccharide also contains two O-acetyl groups in the repeating unit, located on the Hep residue. The repeating structure can be written as: [see text for equation]. This is a novel structure in bacterial cell-wall polysaccharides from Gram-positive bacteria.     

Kinetic resolution of 2-hydroxybutanoate racemic mixtures by NAD-independent L-lactate dehydrogenase

Optically active d-2-hydroxybutanoate is an important building block intermediate for medicines and biodegradable poly(2-hydroxybutanoate). Kinetic resolution of racemic 2-hydroxybutanoate may be a green and desirable alternative for d-2-hydroxybutanoate production. In this work, d-2-hydroxybutanoate at a high concentration (0.197 M) and a high enantiomeric excess (99.1%) was produced by an NAD-independent l-lactate dehydrogenase (l-iLDH) containing biocatalyst. 2-Oxobutanoate, another important intermediate, was co-produced at a high concentration (0.193 M). Using a simple ion exchange process with the macroporous anion exchange resin D301, d-2-hydroxybutanoate was separated from the biotransformation system with a high recovery of 84.7%.